Mitochondrial dysfunction has been implicated in GSD-Ia but the underlying mechanism and its contribution to HCA/HCC development remain unclear. We have shown that hepatic G6Pase-alpha deficiency leads to downregulation of SIRT1 signaling that underlies defective hepatic autophagy in GSD-Ia. SIRT1 is a NAD+-dependent deacetylase that can deacetylate and PGC-1alpha, a master regulator of mitochondrial integrity, biogenesis, and function. We hypothesized that downregulation of hepatic SIRT1 signaling in G6Pase-alpha-deficient livers impairs PGC-1alpha activity, leading to mitochondrial dysfunction. Here we show that the G6Pase-alpha-deficient livers display defective PGC-1alpha signaling, reduced numbers of functional mitochondria, and impaired oxidative phosphorylation. Overexpression of hepatic SIRT1 restores PGC-1alpha activity, normalizes the expression of electron transport chain components, and increases mitochondrial complex IV activity. We have previously shown that restoration of hepatic G6Pase-alpha expression normalized SIRT1 signaling. We now show that restoration of hepatic G6Pase-alpha expression also restores PGC-1alpha activity and mitochondrial function. Finally, we show that HCA/HCC lesions found in G6Pase-alpha-deficient livers contain marked mitochondrial and oxidative DNA damage. Taken together, our study shows that downregulation of hepatic SIRT1/PGC-1alpha signaling underlies mitochondrial dysfunction and that oxidative DNA damage incurred by damaged mitochondria may contribute to HCA/HCC development in GSD-Ia. We have shown that hepatic G6Pase-alpha deficiency-mediated steatosis leads to defective autophagy that is frequently associated with carcinogenesis. We now show that hepatic G6Pase-alpha deficiency also leads to enhancement of hepatic glycolysis and hexose monophosphate shunt (HMS) that can contribute to hepatocarcinogenesis. The enhanced hepatic glycolysis is reflected by increased lactate accumulation, increased expression of many glycolytic enzymes, and elevated expression of c-Myc that stimulates glycolysis. The increased HMS is reflected by increased G6P dehydrogenase activity, elevated production of NADPH, and the reduced glutathione. We show that that restoration of hepatic G6Pase-alpha expression normalizes both glycolysis and HMS in GSD-Ia. Moreover, the HCA/HCC lesions in L-G6pc-/- mice exhibit elevated levels of hexokinase 2 and the M2 isoform of pyruvate kinase which play an important role in aerobic glycolysis and cancer cell proliferation. Taken together, hepatic G6Pase- deficiency causes metabolic reprogramming, leading to enhanced glycolysis and elevated HMS that along with impaired autophagy can contribute to HCA/HCC development in GSD-Ia. The hallmarks of GSD-Ia are impaired glucose homeostasis and long-term risk of HCA/HCC. Currently, there is no therapy to address HCA/HCC in GSD-Ia. We have previously developed a rAAV vector-mediated gene therapy for GSD-Ia and shown that rAAV-G6PC-treated G6pc-/- mice expressing 3% of normal hepatic G6Pase-alpha activity maintain glucose homeostasis and do not develop HCA/HCC. However, it remains unclear whether G6PC gene transfer at the tumor developing stage of GSD-Ia can prevent tumor initiation or abrogate the pre-existing tumors. Using L-G6pc-/- mice that develop HCA/HCC, we now show that treating the mice at the tumor-developing stage with rAAV-G6PC restores hepatic G6Pase-alpha expression, normalizes glucose homeostasis, and prevents de novo HCA/HCC development. The rAAV-G6PC treatment also normalizes defective hepatic autophagy and corrects metabolic abnormalities. However, gene therapy cannot restore G6Pase-alpha expression in the HCA/HCC lesions and fails to abrogate any pre-existing tumors. The major regulators of hepatic glucose metabolism are the glucocorticoids that promote gluconeogenesis. We examined the expression of 11 -hydroxysteroid dehydrogenase type-1 (11HSD1) that mediates local glucocorticoid activation by converting inert cortisone (in human) and dehydrocorticosterone (in rodents) into active cortisol and corticosterone, respectively. We show that the expression of 11HSD1 is down-regulated in HCA/HCC lesions, leading to impairment in glucocorticoid signaling critical for gluconeogenesis activation. This suggests that local glucocorticoid action downregulation in the HCA/HCC lesions may suppress gene therapy mediated G6Pase- restoration. Collectively, our data show that rAAV-mediated gene therapy can prevent de novo HCA/HCC development in L-G6pc-/- mice at the tumor developing stage, but it cannot reduce any pre-existing tumor burden. GSD-Ia is characterized by impaired glucose homeostasis with a hallmark hypoglycemia, following a short fast. Previously, we have developed rAAV vectors expressing either the wild-type (WT) (rAAV-hG6PC-WT) or codon-optimized (co) (rAAV-co-hG6PC) human (h) G6Pase-alpha. We showed that G6pc-/- mice treated with either rAAV-hG6PC-WT or rAAV-co-hG6PC maintain glucose homeostasis and do not develop HCA/HCC if they restore 3% of normal hepatic G6Pase-alpha activity. The codon-optimized vector, which has a higher potency, is currently being used in a phase I/II clinical trial for human GSD-Ia (NCT 03517085). While routinely used in clinical therapies, codon-optimized vectors may not always be optimal. Codon-optimization can impact RNA secondary structure, change RNA/DNA protein binding sites, affect protein conformation and function, and alter post-transcriptional modifications that may reduce potency or efficacy. We therefore sought to develop alternative approaches that could improve the expression yet minimize the impact of sequence changes due to broad codon-optimization. The human, dog, mouse, and rat G6Pase-alpha share 87-91% sequence identity. Intriguingly, in vitro expression assays have routinely shown that the canine G6Pase-alpha isozyme is significantly more active than hG6Pase-alpha. We therefore expanded our analysis to compare G6PC genes across the evolutionary tree, seeking potential codon changes which could enhance enzymatic activity of hG6Pase-alpha. We identified a Ser-298 to Cys-298 substitution naturally found in canine, mouse, rat, and several primate G6Pase-alpha isozymes, that when incorporated hG6Pase-alpha sequence, markedly enhanced enzymatic activity. Using G6pc-/- mice, we show that the efficacy of the rAAV-hG6PC-S298C vector was 3-fold higher than that of the rAAV-hG6PC-WT vector. The rAAV-hG6PC-S298C vector with increased efficacy, that minimizes the potential problems associated with codon-optimization, offers a valuable vector for clinical translation in human GSD-Ia.